Mechanistic Basis of Circadian Clocks in Bmal1 Knockout Mice

Bmal1 基因敲除小鼠生物钟的机制基础

• 批准号: 10798455
• 负责人: Akhilesh Basi Reddy
• 金额: $ 24.5万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10798455
• 关键词: ARNTL gene; Affect; Automobile Driving; Behavioral; Cell physiology; Cells; ChIP-seq; Circadian Dysregulation; Circadian Rhythms; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Diabetes Mellitus; Disease; ETS Family Protein; Family; Feedback; Future; Genes; Genetic Transcription; Genomics; Goals; Health; Heart Diseases; Human; Immunoprecipitation; Knock-out; Knockout Mice; Life; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mental disorders; Metabolic; Metabolic Diseases; Modeling; Molecular; Mus; Nuclear; Oxidation-Reduction; Periodicity; Physiological; Physiology; Play; Process; Proteins; Proteome; Proteomics; Pulmonary Inflammation; Research; Role; Societies; System; Testing; Therapeutic Intervention; Tissues; Transcript; Vascular Diseases; circadian; circadian pacemaker; experimental study; human disease; insight; novel; posttranscriptional; programs; recruit; shift work; transcription factor; transcriptome

PROJECT SUMMARY Circadian clocks are believed to exist at almost all levels of life and play a fundamental role in maintenance of physiological and behavioral processes in accordance with the day-night cycle. The conventional model that describes the circadian clockwork at the molecular level revolves around transcriptional/translational feedback loops (TTFLs). In these models, BMAL1 is believed to act as an indispensable component of the timekeeping system. However, we have found pervasive molecular oscillations in the transcriptome and proteome of Bmal1-/- mice. A research program will be undertaken to obtain a comprehensive mechanistic understanding of these “non-canonical” circadian rhythms in Bmal1-/- mice. The project will broadly focus on understanding transcriptional (Aim 1) and post- transcriptional (Aim 2) functioning of cells and tissues from Bmal1-/- mice. (Aim1) Our preliminary data suggest that the novel circadian rhythms we see might be underpinned by the recruitment of ETS family transcription factors into the clockwork. We will functionally test their role by knocking out key ETS proteins using CRISPR. We will also perform ChIP-seq experiments to find genomic targets driving rhythmic transcripts that we see. In addition, we will perform nuclear proteomics to elucidate novel transcription factors that might mediate rhythmic transcription. Furthermore, we will perform protein interaction analyses using immunoprecipitation mass spectrometry to determine how ETS proteins and redox proteins may physically interact. (Aim 2) We found redox oscillations in Bmal1 knockout cells, implying that these play a role in the clockwork of Bmal1-/- mice. Consequently, we will investigate novel redox oscillations in Bmal1 knockout cells using a novel redox proteomics workflow that we have developed. In addition, we will characterize the rhythmic phospho-proteome and kinome of cells, which we have found could be profoundly affected by deletion of Bmal1. Finally, we will determine whether metabolic circadian oscillations occur in Bmal1 knockouts. Gaining new molecular insights into the circadian clockwork will guide future therapeutic interventions to alleviate the disorders associated with circadian disruption, which are highly prevalent in contemporary society.

项目摘要 生物钟被认为存在于生活的几乎所有层面，并在以下方面发挥着重要作用： 根据昼夜循环维持生理和行为过程。的 在分子水平上描述昼夜节律的传统模型围绕着 转录/翻译反馈环（TTFL）。在这些模型中，BMAL 1被认为是 是计时系统不可或缺的组成部分。然而，我们发现了广泛的分子 Bmal 1-/-小鼠转录组和蛋白质组的振荡。将开展一项研究计划， 为了获得对这些“非规范”昼夜节律的全面的机械理解， Bmal 1-/-小鼠。该项目将广泛关注理解转录（目标1）和后， 转录（目标2）功能的细胞和组织从Bmal 1-/-小鼠。（目标1）我们的初步数据 这表明，我们看到的新的昼夜节律可能是由ETS家族的招募所支撑的。 将转录因子导入发条我们将在功能上测试他们的作用， 使用CRISPR的蛋白质。我们还将进行ChIP-seq实验，以找到驱动基因组的基因组靶点。 我们看到的有节奏的抄本。此外，我们将进行核蛋白质组学，以阐明新的 可能介导节律性转录的转录因子。此外，我们将执行蛋白质 使用免疫沉淀质谱进行相互作用分析，以确定ETS蛋白和 氧化还原蛋白可以物理相互作用。(Aim 2）我们发现Bmal 1敲除细胞中存在氧化还原振荡， 这意味着它们在Bmal 1-/-小鼠的时钟机制中发挥作用。因此，我们将研究新的 使用一种新的氧化还原蛋白质组学工作流程在Bmal 1敲除细胞中进行氧化还原振荡， 开发此外，我们将描述细胞的节律性磷酸化蛋白质组和激酶组， 我们发现Bmal 1基因的缺失会严重影响细胞的功能。最后，我们将确定 代谢昼夜节律振荡发生在Bmal 1敲除中。获得新的分子见解， 昼夜节律将指导未来的治疗干预，以减轻与糖尿病相关的疾病。 昼夜节律紊乱，这在当代社会非常普遍。